DESCRIPTION (provided by candidate): The known neural substrates of sleep and wakefulness are comprised of a diversity of brainstem and forebrain cell groups, including the monoaminergic, cholinergic, GABAergic, and orexinergic systems. However, ablation of these cell groups do not produce more than a 5-10% decrease in total wakefulness, suggesting that there are still undescribed cell groups in the wake-promoting circuitry that were not affected by those lesions. Indeed, the current models of sleep-wake control do not include the location of the brain nuclei nor the neurotransmitter content of the ascending reticular activating system first proposed by Moruzzi and Magoun in the late 1940s. Although long postulated, the contribution of glutamatergic neurons in the brainstem ascending arousal system has not been resolved. The long term objective of the proposed studies is to establish two specific nuclei in the pons as a crucial part of the brainstem reticular formation. The specific aims are to establish the glutamatergic neurons in the pontine parabrachial nucleus and precoeruleus area (PB-PC) as a novel wake-promoting cell population and to determine their ascending pathways to the forebrain and cortex. To test the hypothesis that glutamatergic cells in PB-PC are necessary for normal wakefulness, we will use a combination of genetic and molecular tools to first selectively remove the glutamatergic synaptic transmission from the PB-PC in adult mice and then record their sleep-wake activity using EEG/EMG. We predict that removal of an important wake-promoting glutamatergic system will reduce the amount of wakefulness (thereby increasing the total daily amount of sleep). Using novel adeno-associated viral vectors microinjected in the PB-PC of transgenic mice, we will then elucidate the brain targets of the glutamatergic PB-PC wake-promoting neurons. The rationale for these studies is that the total amount of daily wakefulness depends on the glutamatergic neurotransmission from PB-PC to the basal forebrain, hypothalamus, thalamus, and frontal cortex. These studies use an integrative, systems-based approach to studying the neural basis of wakefulness in mice. These experiments are especially relevant to understanding the mechanisms that control sleep-wake patterns. These data will change the existing models of sleep-wake control by establishing a previously underappreciated brain region (PB-PC) as an important area for the mechanism of wakefulness. Moreover, these results will be directly related to human sleep disorders such as insomnia, narcolepsy, REM behavior disorder, and obstructive sleep apnea.